Project Abstract/Summary. Bacteria, like human beings, often find themselves under stressful circumstances. Bacterial cells, like other living organisms, have evolved stress response pathways to cope with adverse conditions. One of these stress response pathways, called the SOS response, is triggered by DNA damage in bacterial cells. Among other things, the SOS response triggers the expression of alternate DNA polymerases which can repair DNA that has been severely damaged, for example by ultraviolet (UV) light, environmental chemicals, or antibiotics. Repair of damaged DNA, called trans-lesion synthesis, forces the bacteria to use error-prone DNA polymerases, such as DNA polymerases IV and V. As a result, the SOS response triggers an increase in the mutation rate observed in bacteria. The SOS response triggers a strong increase in the rate at which bacteria acquire resistance to antibiotics, as well as mutations in other genes. Zinc blocks the SOS response in bacteria, and prevents the increase in mutation rate, which is also called the mutator response, or hypermutation. Zinc blocks hypermutation by preventing a key bacterial protein, RecA, from binding to single-stranded DNA. RecA is the molecule that serves as the sensor of DNA damage in E. coli and in other bacteria, and which triggers the onset of the SOS response. In addition to triggering an increase in the rate of acquisition of antibiotic resistance within a bacterial strain, our laboratory has recently shown that the SOS response can trigger the transfer of an antibiotic resistance gene between species. Zinc also blocked this horizontal transfer of DNA from Enterobacter cloacae to E. coli. This grant application proposes experiments to complete our understanding of how zinc blocks the function of RecA, and thereby blocks hypermutation as well. The additive or synergistic interaction between zinc and nitric oxide donors will also be explored. In addition, experiments will be conducted in vivo in rabbit intestine to quantitate how much the SOS response contributes to hypermutation, and if it can be blocked by zinc in the gut. The intestinal tract, with its rich microbiome, is often the anatomic site in which antibiotic resistance elements are exchanged among microbes. Currently, our country and world are in the middle of an antibiotic resistance crisis. Understanding the protective effects of zinc could allow us to preserve the remaining antibiotics that are still active against multi-drug resistant bacterial pathogens, and could be used to prevent emergence of resistance against new antibiotics that might be developed in the future.